Immune enhancement

ABSTRACT

A method for stimulation of transcription factor AP (activator protein)-1 by administering a compound of the formula (I)  
                 
 
     wherein  
     R is selected from methyl, ethyl, n-propyl, iso-propyl, c-propyl, n-butyl, sec.-butyl, iso-butyl, tert.-butyl, c-butyl, n-pentyl, sec.-pentyl, iso-pentyl, tert.-pentyl, neo-pentyl, c-pentyl, c-hexyl and c-heptyl; R Na  and R Nb  are the same or different and selected from hydrogen, methyl and ethyl; R 2 , R 3 , R 5  and R 6  are independently selected from hydrogen, methyl, methoxy, thiomethyl, hydroxy, fluoro, chloro, bromo, trifluoromethyl, phenyl and benzyl;  
     n is 1, 2 or 3;  
     R′ and R″ are the same or different and selected from methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec.-butyl, and iso-butyl or R′ and R″ together form a saturated heterocyclic ring of 5-7 atoms; and pharmaceutically acceptable salts, hydrates and solvates thereof, to a mammal in need thereof.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. provisionalapplication No. 60/216,329 filed Jul. 5, 2000.

FIELD OF THE INVENTION

[0002] The present invention relates to substituted benzamides, whichare enhancers of transcription factor AP (activator protein)-1, tocompositions containing them, and to methods for clinical treatment ofdiseases associated with immune suppressive states and to the use of thebenzamides for the preparation of a medicament for stimulation oftranscription factor AP-1. Such compounds are particularly useful in thetreatment of a variety of diseases associated with immune suppressionand low capability to produce IL (interleukin)-2. Such diseases includecancer, autoimmune disease and infectious disease. More particularly,the present invention relates to benzamide derivatives suitable for thetreatment of, for example, solid tumours, rheumatoid arthritis (RA) andAIDS. The compounds of the present invention are also suitable for thetreatment of manic-depressive illness.

BACKGROUND OF THE INVENTION

[0003] AP-1 is a transcriptionally active protein heterodimer containingmembers of the Fos (e.g. c-Fos, FosB, Fra-1, Fra-2) and the Jun (e.g.c-Jun, JunB, JunD) family of proteins. The AP-1 transcription factorsare stimulated by e.g. growth factors, cytokines, T cell activators andneurotransmitters and act as dimers binding to DNA in many promotersincluding proteases and cytokines like IL-2. C-Jun and c-Fos knock-outmice have been produced showing embryonic lethality andosteopetrosis/lymphopenia/behavioural abnormalities, respectively(Johnson et al., 1992). These results emphasise that the AP-1 site ispivotal in many different genes and points out lymphocyte regulation andbehaviour, regulation in the central nervous system, as two distinctbiological hotspots. Therefore, immune suppression with low capabilityto produce IL-2 and behavioural disorders are two very different medicalindication areas where AP-1 activity is suboptimal and where AP-1enhancers can be applied.

[0004] In U.S. Pat. No. 3,177,252 some substituted benzamide derivativesincluding metoclopramide (The Merck Index 12^(th) Ed., entry 6226) aredisclosed as being useful for the treatment of emesis.

[0005] The compound metoclopramide may be associated with depressionand, because of its central as well as peripheral dopamine-blockingproperties, may cause most unwanted tardive dyskinesia.Structure-activity relationship studies have demonstrated the linkbetween the diaminoethylene bridge and the dopamine-D₂ blockade.

[0006] In GB 1,174,956 quaternary ammonium salts of N-substitutedbenzamide derivatives and their action to accelerate the automaticmotility of digestive tract are disclosed.

[0007] In J. Org. Chem. USSR 22, 578-582 (1986), the synthesis ofN-(3-dimethylamino-propyl)-3-nitro-4-acetylaminobenzamide is described.

[0008] In U.S. Pat. No. 4,568,685 someN-[(1H-1,2,4-triazol-1-yl)alkyl]arylamides are disclosed as beinginhibitors of thromboxane synthetase enzyme.

[0009] In U.S. Pat. No. 4,568,687 someN-[ω(1H-imidazole-1-y1)alkyl]arylamides are disclosed as beinginhibitors of thromboxane synthetase enzyme and are also useful in thetreatment of hypertension and myocardial ischemia.

[0010] In Analytical Profiles of Drug Substances vol. 4, K Florey, Ed.(Academic Press, New York, 1975) pp 333-383, procainamide (The MerckIndex 12^(th) Ed., entry 7936) is described as an antiarrhytmic agent.

[0011] We have now discovered a novel method of stimulating thetranscription factor AP-1 using substituted benzamides.

SUMMARY OF THE INVENTION

[0012] Immune Suppression in Cancer, Autoimmune Disease and InfectiousDisease

[0013] Immune system-based approaches for the treatment of malignantdisease have focused on cytolytic effector cells such as cytotoxic Tlymphocytes (CTL), and natural killer (NK) cells. It has also beendemonstrated that tumour-bearing mice can be cured using a wide varietyof approaches, some of which involve IL-2 mediated enhancement of CTLand NK cell activity. However, the apparent success in mice stands incontrast to the current situation in the clinic, wherein only a minorityof patients have thus far benefited from CTL- or NK cell-basedanti-tumour approaches. This is probably a result from tumour-inducedimmune suppression (Whiteside, 1999; Kiessling et al., 1999). One of theunderlying causes of tumour-associated immune suppression of CTL and NKcell activity, the intracellular signalling deficiency resulting fromreduction of the TCR/CD3 zeta chain expression of the T cells, is alsoshared with HIV infection, leprosy, and rheumatoid arthritis. Thissignalling deficiency is overrun by IL-2 treatment in vitro. IL-2 is acentral cytokine in the development of functional immune responses andit has been clearly shown that the AP-1 site of the IL-2 promoter ispivotal for optimal activity (Sundstedt and Dohlsten, 1998). Distinctbenzamides would therefore represent an alternative treatment for immunestimulation and IL-2 enhancement in immune suppressive states ofdisease. In addition, several treatment regiments such as cytostatic andradiation therapy applied in the treatment of cancer result in unwantedimmune suppression, an induced state that would be compensated for byadministering compounds of the present invention.

[0014] Manic-Depressive Illness

[0015] Lithium and sodium valproate (VPA) (The Merck Index 12^(th) Ed.,entry 10049) are effective in the treatment of bipolar disorders(manic-depressive illness) and may function through the regulation ofsignal transduction pathways and transcription factors such as c-Fos andc-Jun, which in turn results to changes in gene expression. Thelong-term efficacy of lithium and VPA in bipolar disorders suggests thatthe regulation of gene expression may be an important target for thesedrugs. These two structurally highly dissimilar agents, lithium and VPA,increase AP-1 DNA binding activity in areas of rodent brain ex vivo andin human neuronal cells in culture (Yuan et al., 1998; Chen et al.,1999). Both treatments also increase the expression of a reporter genedriven by an AP-1-containing promoter, and mutations in the AP-1 sitesof the reporter gene promoter markedly attenuate these effects. Bothtreatments also increase the expression of several endogenous proteins,which genes are known to be regulated by AP-1. These effects suggestthat the temporal regulation of AP-1 mediated gene expression incritical neuronal circuits may play a role in the long-term therapeuticefficacy of lithium and VPA and point out that also other AP-1 enhancerslike distinct benzamides could potentially act as modulators of e.g.manic-depressive illness.

DESCRIPTION OF THE INVENTION

[0016] A primary objective of the present invention is to providebenzamide compounds which by virtue of their pharmacological profile,with high potency in experimental models and low level of side-effects,are considered to be of value in the treatment of disease associatedwith immune suppressive states e.g. for the stimulation, enhancement ormodulation of the immune response. Included in the invention is also theuse of the compounds for the stimulation of transcription factor AP-1.In a particular aspect, this invention provides preparation of amedicament for the stimulation of transcription factor AP-1, a method oftreating diseases in which the disease pathology may be therapeuticallymodified by stimulating AP-1. Examples of such diseases are cancer,autoimmune disease and infectious disease. More particularly, thepresent invention relates to benzamide derivatives suitable for thetreatment of, for example, solid tumours, rheumatoid arthritis (RA) andAIDS. The compounds of the present invention are also suitable for thetreatment of manic-depressive illness.

[0017] The term “treatment” as used herein includes prophylaxis as wellas relieving the symptoms of disease.

[0018] It has now surprisingly been found that the compounds of formula(I)

[0019] wherein

[0020] R is selected from methyl, ethyl, n-propyl, iso-propyl, c-propyl,n-butyl, sec.-butyl, iso-butyl, tert.-butyl, c-butyl, n-pentyl,sec.-pentyl, iso-pentyl, tert.-pentyl, neo-pentyl, c-pentyl, c-hexyl andc-heptyl;

[0021] R_(Na) and R_(Nb) are the same or different and selected fromhydrogen, methyl and ethyl;

[0022] R₂, R₃, R₅ and R₆ are independently selected from hydrogen,methyl, methoxy, thiomethyl, hydroxy, fluoro, chloro, bromo,trifluoromethyl, phenyl and benzyl;

[0023] n is 1, 2 or 3;

[0024] R′ and R″ are the same or different and selected from methyl,ethyl, n-propyl, iso-propyl, n-butyl, sec.-butyl, and iso-butyl or R′and R″ together form a saturated heterocyclic ring of 5-7 atoms havingthe formula

[0025] wherein p is 1, 2, 3;

[0026] X is selected from CHRhet₁, NRhet₁ and O with the proviso that pis 2 or 3 when X is NRhet₁, and O;

[0027] Rhet₁ is selected from hydrogen and C₁₋₅ alkyl, optionallyfunctionalised with OH, halogen (F, Cl and Br), CN, COORhet₂, N(Rhet₂)₂wherein Rhet₂ independently is selected from H, C₁₋₄ alkyl;

[0028] with the proviso that when R′ and R″ are methyl then R cannot bemethyl; and pharmaceutically acceptable salts, hydrates and solvatesthereof; are unexpectedly effective and specific in the treatment ofindividuals suffering from cancer, autoimmune disease, infectiousdisease and manic-depressive illness.

[0029] In a preferred embodiment of the invention

[0030] R is selected from methyl, ethyl, n-propyl and iso-propyl,

[0031] R_(Na) and R_(Nb) are hydrogen,

[0032] one of R₂, R₃, R₅ and R₆ is selected from methyl, methoxy,thiomethyl, hydroxy, fluoro, chloro, trifluoromethyl and phenyl,

[0033] R′ and R″ are selected from methyl, ethyl, n-propyl and n-butylwith the proviso that when R′ and R″ are methyl then R cannot bemethyl;.

[0034] The compounds of formula (I) were assayed for AP-1 enhancement.The compounds of this invention were tested in assays for the modulationof stimulated activity of an AP-1-driven reporter gene in the Jurkat Tcell line. The activation of this reporter gene results in luciferaseproduction and the amounts of produced luciferase parallels the level ofAP-1 activity. A high level of AP-1 activity is pivotal to e.g. highproduction of IL-2.

[0035] All embodiments of the invention as disclosed in the claims areherewith included in the specification.

[0036] The following examples are intended to illustrate the inventionwithout restricting the scope thereof.

[0037] The compounds of formula (I) may be prepared by methods wellknown in the literature. The general solution preparation is shown inScheme 1 and 2.

[0038] A benzamide derivative of formula (I) may be prepared byconventional methods and, for example, by first the reaction of a4-amino-benzoic acid methyl ester (II) with an acid chloride (III) oranhydride in an inert solvent such as dichloromethane in the presence oftriethylamine (Scheme 1). The resulting 4-acylamino-benzoic acid methylesters (IV) and a N,N-substituted alkylenediamine (V) is then condensedin an excess of V in the presence of a catalytic amount ammoniumchloride to form the compounds of formula (I) (Scheme 2). Alternatively,the methylester is first hydrolysed and then activated usingconventional methods, such as ethylchloroformate,dicyclohexylcarbodiimide (DCC) or2-(1H-benzotriazol-1-y1)-1,1,3,3-tetramethyluronium tetrafluoroborat(TBTU). The starting materials used herein are commercially available orare prepared by conventional methods found in standard reference bookssuch as the Compendium of Organic Synthetic Methods, vol. I-VI(Wiley-Interscience), well known to those of ordinary skill in the art.

[0039] Acid addition salts of the compounds of formula (I) are preparedin a standard manner in a suitable solvent and in excess of an acid,such as hydrochloric, hydrobromic, sulphuric, maleic and succinic acid.

EXAMPLE 1 4-iso-Butyrylamino-2-methoxy-benzoic acid methyl ester

[0040] 1.81 g of 4-amino-2-methoxy-benzoic acid methyl ester and 1.4 gof triethylamine were dissolved in 20 ml of dichloromethane. 1.23 g ofisobutyryl chloride in 5 ml of dichloromethane was added dropwise at 0°C. The reaction mixture was allowed to reach room temperature andstirred for 3 hours. The reaction mixture was washed with water, driedand the solvents were evaporated to yield 1.9 g of the title product.

[0041] 1H NMR (CDCl₃): δ1.22 (6H, d), 2.53 (1H, m), 3.83 (3H, s), 3.83(3H, s), 6.85 (1H, d), 7.70 (1H, s), 7.75 (1H, d), 7.84 (1H, s).

EXAMPLE 2N-[(2-Diethylamino)propyl]-4-isobutyrylamino-2-methoxy-benzamide

[0042] 0.3 g of 4-iso-butyrylamino-2-methoxy-benzoic acid methyl esterwas dissolved in 3 ml of N,N-diethyl-propylenediamine together with acatalytic amount of ammonium chloride. The reaction mixture was refluxedfor 3 hours. Dichloromethane was added and washing 4 times with waterremoved excess diamine. Drying and evaporation of the solvents yielded0.15 g of the title compound.

[0043] 1H NMR (CDCl₃): δ0.97 (6H, t), 1.20 (6H, d), 1.71 (2H, m), 2.48(6H, m), 2.59 (1H, m), 2.46 (2H, q), 3.90 (3H, s), 6.80 (1H, d), 7.94(1H, s), 8.03 (1H, d), 8.29 (1H, s).

EXAMPLE 3N-[3-(Diethylamino)propyl]-4-iso-buturylamino-3-hydroxy-benzamide

[0044] 4-iso-Butyrylamino-3-hydroxy-benzoic acid (0.089 g) and TBTU(0.128 g) in chloroform (15 ml) was stirred for 2 hours.N,N-Diethyl-propylenediamine (0.052 g) was added and the mixture wasstirred for 1 hour. Evaporation of the solvent gave a residue, which waschromatographed on a silica gel column first eluted/washed withethylacetate and then eluted with ethylacetate-methanol-triethylamine(12:4:1) to yield 0.03 g, of the title product.

[0045] 1H NMR (CDCl₃): δ1.14 (t, 6H) 1.25 (m, 6H) 2.01 (m, 2H) 2.61 (m,1H) 2.98 (m, 2H) 3.04 (m, 2H) 3.44 (m, 2H) 7.35 (m, 1H) 7.80 (d, 1H)7.92 (m, 1H) 8.21 (d, 1H) 8.36 (s, 1H).

EXAMPLE 4 4-(N-iso-Butyryl-N-methylamino)-benzoic acid

[0046] 4-Methylamino-benzoic acid methyl ester (0.41 g) were suspendedin chloroform (20 ml). Isobutyryl chloride (0.79 g) in chloroform (10ml) was added dropwise for 30 minutes and then dropwise triethylamine (1ml) dissolved in chloroform (5 ml). The reaction mixture was stirred for3 hours and the solvent was evaporated. 1M NaOII(aq) (40 ml) was addedand the mixture stirred overnight. The solution was filtered to removeunsolved material and then acidified with 2M HCl(aq). Filtration anddrying in vacuum yielded 0.48 g of the title product.

EXAMPLE 5N-[3-(Diethylamino)propyl]-4-(N-iso-butyryl-N-methylamino)-benzamide

[0047] To a stirred solution of 4-(iso-butyryl-N-methylamino)-benzoicacid (0.088 g) and triethylamine (0.041 g) in chloroform (4 ml) wasadded a solution of ethyl chlorformate (0.049 g) in chloroform (1 ml).The mixture was stirred under an N₂ atmosphere at room temperature for1½ hours and then cooled to 0° C. N,N-Diethyl-propylenediamine (0.051 g)in chloroform (1 ml) was added and the mixture was stirred over night.The mixture were washed with 0.5 M NaOH(aq) and water. Drying andevaporation of the solvent gave a residue, which was chromatographed ona silica gel column first eluted/washed with ethylacetate and theneluted with ethylacetate-methanol-triethylamine (12:4:1) to yield 0.098g, of the title product.

[0048] 1H NMR (CDCl₃): δ1.01 (t, 6H) 1.04 (t, 6H) 1.77 (m, 2H) 2.48 (m,1H) 2.61 (m, 6H) 3.23 (s, 3H) 3.57 (m, 2H) 7.21 (m, 1H) 7.83 (m, 1H)8.80 (s, 1H).

[0049] Examples 6 to 12 were prepared by the method described in Example5.

EXAMPLE 6N-[3-(Diethylamino)propyl]-4-iso-butyrylamino-3-methoxy-benzamide

[0050] 1NMR (CDCl₃): δ1.07 (t, 6H) 1.25 (d, 6H) 1.80 (m, 2H) 2.55 (m,1H) 2.64 (m, 6H) 3.56 (m, 2H) 3.94 (s, 3H) 7.27 (m, 1H) 7.55 (d, 1H)7.91 (s, 1H) 8.43 (d, 1H) 8.70 (s, 1H).

EXAMPLE 7N-[3-(Diethylamino)propyl]-4-(iso-butyryl-N-methylamino)-2-methoxy-benzamide

[0051] 1H NMR (CDCl₃): δ1.04 (m, 12H) 1.80 (m, 2H) 2.56 (m, 7H) 3.24 (s,3H) 6.75 (s, 1H) 6.88 (m, 1H) 7.99 (m, 1H) 8.17 (d, 1H).

EXAMPLE 8 N-(3-morpholinopropyl)-4-iso-butyrylamino-2-methoxy-benzamide

[0052] 1H NMR (CDCl₃): δ1.24 (d, 6H) 1.86 (m, 2H) 2.52 (m, 7H) 3.51 (m,2H) 3.75 (m, 4H) 3.97 (s, 3H) 6.71 (m, 1H) 7.37 (s, 1H) 7.94 (s, 1H)8.09 (d, 1H).

EXAMPLE 9N-[4-(Dimethylamino)butyl]-4-iso-butyrylamino-2-methoxy-benzamide

[0053] 1H NMR (CDCl₃): δ1.25 (d, 6H) 1.65 (m, 4H) 2.37 (m, 6H) 2.52 (m,3H) 3.45 (m, 2H) 3.97 (s, 3H) 6.73 (m, 1H) 7.43 (s, 1H) 7.88 (m, 1H)7.92 (s, 1H) 8.09 (d, 1H).

EXAMPLE 10N-[3-(4-methylpiperazino)-propyl]-4-iso-butyrylamino-2-methoxy-benzamide

[0054] 1H NMR (CDCl₃): δ1.25 (d, 6H) 1.83 (m, 3H) 2.35 (s, 3H) 2.52 (m,3H) 2.60 (m, 7H) 3.49 (m, 2H) 3.96 (s, 3H) 6.71 (m, 1H) 7.37 (s, 1H)7.94 (m, 2H) 8.08 (d, 1H).

EXAMPLE 11N-[3-(Diethylamino)propyl]-4-iso-butyrylamino-3,5-dichloro-benzamide

[0055] 1H NMR (CDCl₃): δ1.10 (t, 6H) 1.29 (d, 6H) 1.82 (m, 2H) 2.69 (m,7H) 3.55 (m, 2H) 7.03 (s, 1H) 7.82 (s, 2H) 9.22 (s, 1H).

[0056] Pharmacological Methods

[0057] Cells from the Jurkat T cell line were transfected with anAP-1-driven luciferase gene reporter construct (Parra et al., 1997)together with a selection gene vector. Selected clones from theresulting stable AP-1 reporter gene transfectants were used in theassays for AP-1 transcription factor activity. These transfected Jurkatcells (Jurkat/AP-1rep) were grown in RPMI 1640 supplemented withglutamine, hepes, sodium pyruvate, gentamicin, 10% FCS and G418. Toevaluate distinct compounds for their capacity to enhance the AP-1activity, Jurkat/AP-1rep cells were stimulated for 5.5 h in thetemperature of 37° C. with phorbol myristate acetate and ionomycin inthe absence and presence of test compounds. The 96 well platescontaining the cell cultures were then put on ice until harvested. Thesupernatants were removed and the cells were lysed. AP-1 activity wasmeasured as luminiscence produced by luciferase substrate, which wasadded to the wells in conjunction with measurement.

[0058] Superantigen responsive mice were treated with Staphylococcalenterotoxin A in accordance with Belfrage et al. (Belfrage et al. 1995,1997a, 1997b). Plasma and splenocytes were collected at different timepoints to evaluate the induced activity of T cells with and withouttreatment with the compounds of formula (I). It is shown that T cellactivity as measured as IL-2 production, cytotoxic T cell activity andanergy induction (Belfrage et al. 1995, 1997a, 1997b) were modulated bythe treatment.

[0059] Among preferred compounds isN-[(2-diethylamino)propyl]-4-isobutyrylamino-2-methoxybenzamide,hereinafter called Compound A. Compound A was shown to enhance theactivity of the AP-1 driven reporter in a dose dependent fashion down toμM concentrations (FIG. 1 +L).

[0060] FIG. 1 +L. Percentage enhancement of the reporter gene activityin the assay utilising Jurkat cells with a transfected AP-1-drivenluciferase gene reporter.

[0061] The compounds of formula (I) are useful as enhancers of AP-1. Thepresent invention provides useful compositions and formulations of saidcompounds including pharmaceutical compositions and formulations of saidcompounds.

[0062] Effective quantities of the compounds of formula (I) arepreferably administered to a patient in need of such treatment accordingto usual routes of administration and formulated in usual pharmaceuticalcompositions comprising an effective amount of the active ingredient anda suitable pharmaceutically acceptable carrier. Such compositions maytake a variety of forms, e.g. solutions, suspensions, emulsions,tablets, capsules, and powders prepared for oral administration,aerosols for inhalations, sterile solutions for parental administration,suppositories for rectal administration or suitable topicalformulations. Conventional procedures for the selection and preparationof suitable pharmaceutical formulations are described, for example, in“Pharmaceuticals-The Science of Dosage Form Design”, M. B. Aulton,Churchill Livingstone, 1988.

[0063] A suitable daily dose for use in the treatment of disease withassociated immune suppression or manic-depressive illness iscontemplated to vary between 0.0005 mg/kg to about 10 mg/kg body weight,in particular between 0.005 mg/kg to 1 mg/kg body weight, depending uponthe specific condition to be treated, the age and weight of the specificpatient, and the specific patient's response to the medication. Theexact individual dosage, as well as the daily dosage, will be determinedaccording to standard medical principles under the direction of aphysician.

[0064] Various additives to enhance the stability or ease ofadministration of the drug are contemplated. The pharmaceuticalcomposition may also contain additional therapeutically usefulsubstances other than a compound of formula (I).

[0065] The ability of the compounds of the present invention to enhancethe AP-1 activity is clearly evidenced by their ability to enhance thereporter gene activity (see FIG. 1 +L). No unacceptable toxicologicaleffects are expected such as tardive dyskinesia when compounds of thepresent invention are administered in accordance with the presentinvention.

[0066] References

[0067] Belfrage H, Dohlsten M, Hedlund G, Kalland T. 1995 Enhanced andprolonged efficacy of superantigen-induced cytotoxic T lymphocyteactivity by interleukin-2 in vivo. Cancer Immunol Immunother 1995August; 41(2):87-94

[0068] Belfrage H, Dohlsten M, Hedlund G, Kalland T. 1997a Prevention ofsuperantigen-induced down-regulation of T-cell mediated cytotoxicactivity by IL-2 in vivo. Immunology 1997 February; 90(2): 183-8

[0069] Belfrage H, Dohlsten M, Hedlund G, Kalland T. 1997b Prevention ofsuperantigen-induced tolerance in vivo by interleukin-2 treatment.Cancer Immunol Immunother 1997 April; 44(2): 77-82

[0070] Chen G, Yuan P X, Jiang Y M, Huang L D, Manji H K. Valproaterobustly enhances AP-1 mediated gene expression. Brain Res Mol Brain Res1999 January 22; 64(1):52-8

[0071] Johnson R S, Spiegelman B M, Papaioannou V. Pleiotropic effectsof a null mutation in the c-fos proto-oncogene. Cell 1992 November13;71(4):577-86

[0072] Kiessling R, Wasserman K, Horiguchi S, Kono K, Sjoberg J, Pisa P,Petersson M. Tumor-induced immune dysfunction. Cancer Immunol Immunother1999 October; 48(7):353-62

[0073] Parra E, Varga M, Hedlund G, Kalland T, Dohlsten M. Costimulationby B7-1 and LFA-3 targets distinct nuclear factors that bind to theinterleukin-2 promoter: B7-1 negatively regulates LFA-3-induced NF-ATDNA binding. Mol Cell Biol 1997 March; 17(3): 1314-23

[0074] Sundstedt A, Dohlsten M. In vivo anergized CD4+ T cells havedefective expression and function of the activating protein-1transcription factor. J Immunol Dec. 1, 1998;161(11): 5930-6

[0075] Whiteside T L. Signaling defects in T lymphocytes of patientswith malignancy. Cancer Immunol immunother 1999 October; 48(7):346-52

[0076] Yuan P X, Chen G, Huang L D, Manji H K. Lithium stimulates geneexpression through the AP-1 transcription factor pathway. Brain Res MolBrain Res July 15, 1998; 58(1-2):225-30

We claim:
 1. A method for stimulation of transcription factor AP(activator protein)-1 by administering a compound of the formula (I)

wherein R is selected from methyl, ethyl, n-propyl, iso-propyl,c-propyl, n-butyl, sec.-butyl, iso-butyl, tert.-butyl, c-butyl,n-pentyl, sec.-pentyl, iso-pentyl, tert.-pentyl, neo-pentyl, c-pentyl,c-hexyl and c-heptyl; R_(Na) and R_(Nb) are the same or different andselected from hydrogen, methyl and ethyl; R₂, R₃, R₅ and R₆ areindependently selected from hydrogen, methyl, methoxy, thiomethyl,hydroxy, fluoro, chloro, bromo, trifluoromethyl, phenyl and benzyl; n is1, 2 or 3; R′ and R″ are the same or different and selected from methyl,ethyl, n-propyl, iso-propyl, n-butyl, sec.-butyl, and iso-butyl or R′and R″ together form a saturated heterocyclic ring of 5-7 atoms havingthe formula

 wherein p is 1, 2, 3; X is selected from CHRhet₁, NRhet₁ and O with theproviso that p is 2 or 3 when X is NRhet₁ and O; Rhet₁ is selected fromhydrogen and C₁₋₅ alkyl, optionally functionalised with OH, halogen (F,Cl and Br), CN, COORhet₂, N(Rhet₂)₂ wherein Rhet₂ independently isselected from H, C₁₋₄ alkyl; and pharmaceutically acceptable salts,hydrates and solvates thereof, to a mammal in need thereof.
 2. Methodaccording to claim 1 for stimulation, enhancement or modulation of theimmune response by administering compounds of the formula (I).
 3. Methodaccording to claim 2 for the treatment of cancer and unwanted immunesuppression induced by e.g. cytostatic and radiation therapy byadministering compounds of the formula (I).
 4. Method according to claim2 for the treatment of autoimmune disease by administering compounds ofthe formula (I).
 5. Method according to claim 2 for the treatment ofinfectious disease by administering compounds of the formula (I). 6.Methods according to claim 1 for the treatment of manic-depressiveillness by administering of compounds of the formula (I).
 7. Methodaccording to claim 1 by administeringN-[(2-diethylamino)propyl]-4-isobutyrylamino-2-methoxy-benzamide. 8.Method according to claim 1 by administering a compound in a dailydosage of between 0.0005 mg/kg to about 10 mg/kg body weight, inparticular between 0.005 to 1 mg/kg body weight.
 9. Compounds of formula(I)

wherein R is selected from methyl, ethyl, n-propyl, iso-propyl,c-propyl, n-butyl, sec.-butyl, iso-butyl, tert.-butyl, c-butyl,n-pentyl, sec.-pentyl, iso-pentyl, tert.-pentyl, neo-pentyl, c-pentyl,c-hexyl and c-heptyl; R_(Na) and R_(Nb) are the same or different andselected from hydrogen, methyl and ethyl; R₂, R₃, R₅ and R₆ areindependently selected from hydrogen, methyl, methoxy, thiomethyl,hydroxy, fluoro, chloro, bromo, trifluoromethyl, phenyl and benzyl; n is1, 2 or 3; R′ and R″ are the same or different and selected from methyl,ethyl, n-propyl, iso-propyl, n-butyl, sec.-butyl, and iso-butyl or R′and R″ together form a saturated heterocyclic ring of 5-7 atoms havingthe formula

 wherein p is 1, 2, 3; X is selected from CHRhet₁, NRhet₁ and O with theproviso that p is 2 or 3 when X is NRhet₁ and O; Rhet₁ is selected fromhydrogen and C₁₋₅ alkyl, optionally functionalised with OH, halogen (F,Cl and Br), CN, COORhet₂, N(Rhet₂)₂ wherein Rhet₂ independently isselected from H, C₁₋₄ alkyl; with the proviso that when R′ and R″ aremethyl then R cannot be methyl; and pharmaceutically acceptable salts,hydrates and solvates thereof.
 10. Compounds according to claim 9wherein R is selected from methyl, ethyl, n-propyl and iso-propyl,R_(Na) and R_(Nb) are hydrogen, one of R₂, R₃, R₅ and R₆ is selectedfrom methyl, methoxy, thiomethyl, hydroxy, fluoro, chloro,trifluoromethyl and phenyl, with the proviso that when R′ and R″ aremethyl then R cannot be methyl; and R′ and R″ are selected from methyl,ethyl, n-propyl and n-butyl. 11.N-[(2-Diethylamino)propyl]-4-isobutyrylamino-2-methoxy-benzamideaccording to claims 9 and
 10. 12. Compounds of the formula (I)

wherein R is selected from methyl, ethyl, n-propyl, iso-propyl,c-propyl, n-butyl, sec.-butyl, iso-butyl, tert.-butyl, c-butyl,n-pentyl, sec.-pentyl, iso-pentyl, tert.-pentyl, neo-pentyl, c-pentyl,c-hexyl and c-heptyl; R_(Na) and R_(Nb) are the same or different andselected from hydrogen, methyl and ethyl; R₂, R₃, R₅ and R₆ areindependently selected from hydrogen, methyl, methoxy, thiomethyl,hydroxy, fluoro, chloro, bromo, trifluoromethyl, phenyl and benzyl; n is1, 2 or 3; R′ and R″ are the same or different and selected from methyl,ethyl, n-propyl, iso-propyl, n-butyl, sec.-butyl, and iso-butyl or R′and R″ together form a saturated heterocyclic ring of 5-7 atoms havingthe formula

 wherein p is 1, 2, 3; X is selected from CHRhet₁, NRhet₁ and O with theproviso that p is 2 or 3 when X is NRhet₁ and O; Rhet₁ is selected fromhydrogen and C₁₋₅ alkyl, optionally functionalised with OH, halogen (F,Cl and Br), CN, COORhet₂, N(Rhet₂)₂ wherein Rhet₂ independently isselected from H, C₁₋₄ alkyl; with the proviso that when R′ and R″ aremethyl then R cannot be methyl; and pharmaceutically acceptable salts,hydrates and solvates thereof, for therapeutic use.